Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
For most eukaryotic mRNAs, translation initiates with the binding of the cap binding protein to the mRNA cap structure. This is then followed by the binding of several other translation factors, as well as the 43S ribosome pre-initiation complex. This complex travels down the 5′ region of the mRNA molecule while scanning for an initiation codon in an appropriate sequence context. Once the initiation codon has been found and with the addition of the 60S ribosomal subunit, the complete 80S initiation complex initiates protein translation (1,2,3). A second class of mRNAs have been identified which possess translation initiation features different from those described above. Translation from these mRNAs initiates in a cap-independent manner and is believed to initiate with the ribosome binding to internal portions of the leader sequence (4,5,6,7). Most 5′ untranslated leader sequences are very A,U rich and are predicted to lack any significant secondary structure. One of the early steps in translation initiation is the relaxing or unwinding of the secondary mRNA structure (6). Messenger RNA leader sequences with negligible secondary structure may not require this additional unwinding step and may, therefore, be more accessible to the translation initiation components. The ability of a leader sequence to interact with translational components may play a key role in affecting levels of subsequent gene expression.
In work leading up to the present invention, the inventors sought to identify 5′ leader sequences which might be responsible for the regulation of gene expression. The gene initially chosen for investigation was the GLI1 gene.
The gene GLI1 was originally isolated as a highly amplified gene in a malignant glioma (8) and subsequently implicated in the development of other tumor types, including liposarcoma, rhabdomyosarcoma, osteosarcoma and astrocytoma (9,10). It has been shown that GLI1 encodes a transcription factor which is a downstream nuclear component of the Sonic Hedgehog/Patched (SHH/PTC) signalling pathway (11,12,13). This pathway is evolutionarily conserved and found to operate in a number of tissues during vertebrate development and especially in regions involving mesoderm-ectoderm interactions (14,15,16,17). Intercellular signalling by this pathway is initiated when SHH (a secreted protein) binds to PTC (a cell-surface transmembrane protein), resulting in the activation of GLI1 in the nucleus and subsequent expression of target genes. Over-expression of SHH has been shown to upregulate GLI1 in the chick limb buds and in the epidermal ectoderm of frog embryos (18,19) whereas, GLI1 expression is undetectable in SHH null embryos (20,21), confirming that SHH signalling regulates GLI1 expression.
The discovery of PTC mutations in familial and sporadic forms of basal cell carcinoma (BCC), the most common skin cancer, has associated aberrant signalling of the SHH/PTC pathway with the formation of these tumors (22,23,24). The genetic data are supported by experimental evidence showing that over-expression of SHH and other components of this pathway results in the induction of BCCs in transgenic mice and transgenic human skin (25,26,27). Over-expression of GLI1 produces BCC-like lesions in transgenic tadpoles (28) and transforms rodent epithelial cells in cooperation with adenovirus EIA (29) indicating that unregulated expression of GLI1 is oncogenic. Studies have shown that GLI1 expression is greatly increased in BCCs but not in the surrounding normal tissue consistent with a central role in tumor formation (28,30,31).
In addition to GLI1, two other isoforms have been identified in vertebrates (termed •GLI2• and •GLI3•), each encoded by a separate gene (15,17). The GLI genes are highly expressed during development and their expression profiles correlate with organogenesis but show only low level expression in most adult tissues (14,16,17). In the skin, GLI1 expression is readily observed in the epidermal compartment of the developing hair follicle whereas GLI2 and GLI3 transcripts were detected in the surrounding mesenchyme (17,28,31). The role of each GLI in mediating the SHH signal is not yet clear but recent gene ablation studies on GLI2 and GLI3 have shown overlapping roles and indicated some functional redundancy (32). A number of studies have indicated that GLI1 encodes a transcriptional activator, whereas GLI2 and GLI3 encode factors which can act as both an activator or a repressor depending on specific post-translational modifications (33,34,35). Interestingly, GLI2 and GLI3 are now thought to regulate GLI1 transcription directly by binding to the GLI1 promoter (33,35).
In accordance with the present invention, the subject inventors have now identified alternative 5′UTRs of GLI1 transcripts in mammalian tissues which are generated by exon skipping and which confer marked differences in translation efficiency. The inventors• results indicate that post-transcriptional regulation of GLI1 is mediated by the 5′UTR generated through exon skipping and show an association of the most efficiently translated 5′UTR transcript with BCC and cellular proliferation.
More particularly, the inventors surprisingly determined that by altering the number of sequence elements corresponding to pseudo-translation initiation sites, i.e. RUG or RTG triplets (where R is A or G), within the leader sequence of a nucleic acid molecule and prior to the authentic translation initiation site alone or in combination with termination signals in the 5′ UTR or in a different reading frame within the coding sequence and/or in the 3′ UTR, it is possible to modulate gene expression. In a further determination, the present inventors identified that altering nucleotide sequences proximal to the RUG or RTG triplets within a leader sequence also permitted the regulation of gene expression. Thus, the subject inventors have developed a method for the regulation of gene expression in eukaryotic cells including animal cells and plant cells. Furthermore, the identification of expression modulating sequences enables determination to be made as to expected levels of gene expression such as in certain disease conditions or to express traits at selective levels in animal and plant cells.